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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Endotoxin Activity Assay (EAA)
<ol>
	<li>Collect patient blood samples in sterile tubes containing EDTA anticoagulant. Store blood samples at room temperature</li>
	<li>Prepare the EA test tubes for each patient&#39;s blood sample you need to test. Put the tubes in tube racks. Then remove the caps.</li>
	<li>Each EA test consists of 5 different kinds of tubes; use each one for a different portion of the test (refer next steps).
	<ol>
		<li>Use tube #1 (the &#34;Control&#34; tube) to measure the basal activity of the non-specific oxidative burst of patient&#39;s neutrophils in the absence of a specific antibody.</li>
		<li>Use tube #2 (the &#34;Sample&#34; tube) to measure the oxidative burst in response to the Lipopolysaccharide&#160;(LPS)-antibody complex.</li>
		<li>Use tube #3 (the &#34;Max&#34; tube) to measure the maximal oxidative burst of patient&#39;s neutrophils in response to an excess of endotoxin.</li>
		<li>Use tube #4 (the &#34;LPS&#34; tube) as a source of exogenous endotoxin.</li>
		<li>Use tube #5 (&#34;Aliquot&#34; tube) for blood storage. 
		NOTE:&#160;Duplicates of tubes #1, #2, and #3 are provided for a total of 8 tubes to be used for each blood sample being tested (the EA reagent bottle and quality control test can be used for all the tests contained in a pouch).</li>
	</ol>
	</li>
	<li>Using a combi-pipette, pipette a 1 mL volume of the EA reagent from the bottle into tubes #1 (Control tube), #2 (Sample tube), and #3 (Max tube), each one in duplicate. 
	NOTE:&#160;Pipette down the side of the tube to avoid solution splashing back up.</li>
	<li>Mix the patient blood sample by gently inverting the blood collection tube 20 times. Then, pipette 0.5 mL of patient blood into tube #4 (LPS max tube) and tube #5 (Aliquot tube). Vortex tube #4 for 10 s.</li>
	<li>Put the tube racks with all the EA test tubes in the incubator shaker. Close the lid and incubate for 10 min at the temperature of 37&#176;C.</li>
	<li>Open the lid and remove the tube racks from the incubator shaker. Vortex tube #5 (Aliquot tube). Using a sterile tip, pipette 40 &#181;L of blood into tubes #1 and #2, in duplicate.</li>
	<li>Vortex tube #4 (LPS tube). Using the same pipette tip, pipette 40 &#181;L of blood from tube #4 into tube #3 (Max tube), in duplicate.</li>
	<li>Vortex the six final test tubes (#1, #2, #3, and respective duplicates), then place them back into their racks. 
	NOTE:&#160;Ensure that all the tubes are vortexed for the same amount of time.</li>
	<li>Put the tube racks back into the incubating shaker and close the lid. Set the incubating shaker at 100 rpm, then start the motion for 14 min.</li>
	<li>Insert the EA-labeled chip card in the chemiluminometer and press start. After the 14 min incubation, follow the instructions displayed on the chemiluminometer to read the EA tubes in the correct order.</li>
	<li>Gently vortex each tube for 10 s before placing it onto the sample holder of the chemiluminometer. Open the sample drawer and place tube #1 in the sample holder. Then, close the sample drawer and wait for the Relative Light Unit (RLU) reading.</li>
	<li>Repeat step 1.12 for tube 2 and tube 3.</li>
	<li>Repeat step 1.12 for duplicate tubes 1, 2, and 3. 
	NOTE:&#160;Try to vortex all tubes for the same amount of time during steps 1.12&#8211;1.14.</li>
	<li>After all the tubes have been processed, note that the EA results will be calculated and printed automatically. Levels are expressed as EA units and represent the mean of duplicate determinations from the same samples.</li>
	<li>Repeat steps 1.4 to 1.15 for every blood sample that needs to be tested.</li>
	<li>Once the assay has been completed, store the remaining test tubes and EA reagents at 2&#8211;8 &#176;C for up to 30 days.</li>
</ol>

<table><tbody><tr><td>EAA kit</td><td>Spectral Medical Inc.</td><td>EAAST-20</td><td>Package with 20 tests + 1 quality control</td></tr><tr><td>Smart Line TL</td><td>Berthold</td><td>EAASL</td><td>Luminometer</td></tr><tr><td>Incubator shaker</td><td>GRANT</td><td>ES-20</td><td>Mini-incubator shaker</td></tr><tr><td>Vortexer</td><td>VWR</td><td>444-2790</td><td>Vortex instrument</td></tr></tbody></table>

endotoxin activity assay: a chemiluminescence-based bioassay for investigating endotoxin activity in blood sample

Source: Pinciroli, R. et al. <a href="https://www.jove.com/v/58507">Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients.</a> J. Vis. Exp. (2019).
This video demonstrates a rapid bioassay for investigating endotoxin activity in a whole blood sample via a chemiluminescence-based method. This assay is a rapid and sensitive biomarker test for early-stage diagnosis of endotoxin-mediated sepsis.

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

Source: Pinciroli, R. et al. Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients. J. Vis. Exp. (2019).
This ...

In Gram-negative bacteria, lipopolysaccharide endotoxins are vital components of the bacterial outer membrane.
Structurally, an endotoxin is comprised of the O-antigen - outermost polysaccharide chain connected via a central core to lipid A, an immunostimulatory glycolipid crucial for endotoxin activity.
To investigate endotoxin activity, take a test tube with a suitable buffer containing anti-lipid A antibodies, zymosan - an exogenous neutrophil activator, and luminol - a chemiluminescent agent.
Suspend an optimum volume of an infected blood sample comprising endotoxins and blood cells, including neutrophils. Incubate the tube under physiological conditions.
During incubation, anti-lipid A antibody recognizes the lipid-A component of the endotoxin and binds to it via antigen-binding domains, resulting in the formation of an antibody-endotoxin complex.
 The endotoxin-bound antibody&#39;s FC domain binds to the neutrophils&#39; complementary receptor, presenting the attached endotoxins to neutrophils. This binding acts as a priming stimulus for neutrophils, triggering their activation.
Activated neutrophils engulf the zymosan and initiate an immune response, resulting in a rapid release of reactive oxygen species, ROS, from neutrophils. These ROS facilitate luminol oxidation, resulting in light emission by chemiluminescence.
The chemiluminescence signal intensity corresponds to the endotoxin activity in the blood sample.

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Research

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Biological Techniques

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269 ビュー. 出典:Pinciroli, R. et al.重篤な患者における全血エンドトキシン血症の検出のためのエンドトキシン活性アッセイ。J. Vis. Exp.(2019年)。 このビデオは、化学発光ベースの方法により全血サンプル中のエンドトキシン活性を調査するための迅速なバイオアッセイを示しています。このアッセイは、エンドトキシン媒介性敗血症の早期診断のための迅速で高感度なバイオマーカー検...

ビデオ: エンドトキシン活性アッセイ:血液サンプル中のエンドトキシン活性を調査するための化学発光ベースのバイオアッセイ - 概念

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

グラム陰性菌からの単離とリピドAの化学特性評価

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

JoVE Journal

化学

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

健康なヒトにおける静脈内毒素チャレンジ：実験プラットフォームが調査し、全身性炎症を調節するために

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

医学

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

ラットにおける再現可能な集中治療ユニット指向エンドトキシンモデル

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

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Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample